WO1992003456A1 - Oligonucleotides for modulating the effects of cytomegalovirus infections - Google Patents

Oligonucleotides for modulating the effects of cytomegalovirus infections Download PDF

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Publication number
WO1992003456A1
WO1992003456A1 PCT/US1991/005815 US9105815W WO9203456A1 WO 1992003456 A1 WO1992003456 A1 WO 1992003456A1 US 9105815 W US9105815 W US 9105815W WO 9203456 A1 WO9203456 A1 WO 9203456A1
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Prior art keywords
oligonucleotide
gcg
analog
ccg
ccc
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PCT/US1991/005815
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French (fr)
Inventor
Kevin P. Anderson
Kenneth G. Draper
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Isis Pharmaceuticals, Inc.
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Priority to AU84393/91A priority Critical patent/AU649717B2/en
Priority to DE69126710T priority patent/DE69126710T2/en
Priority to CA002089666A priority patent/CA2089666C/en
Priority to KR1019930700439A priority patent/KR970005273B1/en
Priority to US07/927,506 priority patent/US5591720A/en
Priority to EP91914366A priority patent/EP0544713B1/en
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to BR919106751A priority patent/BR9106751A/en
Publication of WO1992003456A1 publication Critical patent/WO1992003456A1/en
Priority to NO93930515A priority patent/NO930515L/en
Priority to FI930658A priority patent/FI930658A/en
Priority to US08/233,711 priority patent/US5595978A/en
Priority to GR970402519T priority patent/GR3024873T3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C12N15/1133—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against herpetoviridae, e.g. HSV
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to the design and synthesis of antisense oligonucleotides which can be administered to inhibit the replication of cytomegalovirus and treat cytomegalovirus infections. These compounds can be used either prophylactically or therapeutically to reduce the severity of disease caused by cytomegaloviruses. Oligonucleotides and oligonucleotide analogs which are specifically hybridizable with RNA targets are described. BACKGROUND OF THE INVENTION
  • Cytomegaloviruses are ubiquitous in nature and are the most common causes of intrauterine infection. Congenital infection is common in newborns of infected mothers. In some populations, as much as 10% of children display perinatal infections. In a small
  • Cytomegalovirus retinitis is a severe problem in immunosuppressed patients that often leads to blindness. Immunosuppressed patients are also very susceptible to CMV pneumonitis, which is one of the most lethal of human viral diseases.
  • CMV pneumonitis which is one of the most lethal of human viral diseases.
  • cytomegalovirus may play a role in the progression of HIV infection to AIDS by stimulating the transcription of the HIV long terminal repeats (LTR) in non-transformed co-infected T cells, histologic examination of adrenals and brains from AIDS patients has suggested that the adrenalitis, encephalitis and peripheral
  • neuropathy were caused by CMV infection.
  • CMV is considered to be an oncogenic virus.
  • CMV can transform cells and stimulate growth. Both human and non-human cells can undergo transformation when incubated with CMV. Transformed cells contain CMV antigens that are oncogenic when inoculated into appropriate
  • Human CMV is a large, enveloped herpesvirus whose genome consists of a double-stranded DNA molecule which is approximately 240,000 nucleotides in length. This genome is the most complex of all DNA viruses and is approximately 50% larger than the genome of herpes simplex virus (HSV). Intact viral DNA is composed of contiguous long (L) and short (S) segments, each of which contains regions of unique DNA sequence flanked by homologous regions of repetitive sequence. As a group, the human CMV isolates share at least 80% sequence homology, making it nearly impossible to classify cytomegaloviruses into subgroups or subtypes, although variations in the restriction
  • CMV gene In permissive human fibroblasts, CMV gene
  • CMV gene expression is regulated by a cascade of genetic events that act at both the transcriptional and translational levels.
  • CMV gene expression can be divided into three phases which resemble those of HSV defined as the immediate early (IE), early and late periods.
  • IE immediate early messenger RNA
  • IE mRNAs immediate early messenger RNAs
  • IE mRNAs synthesized from IE mRNAs; of these, one is a glycoprotein.
  • the IE1 and IE2 proteins are transcriptional activating factors for other CMV genes and the IE3 protein encompasses a region of the CMV genome which can transform NIH 3T3 cells in vitro.
  • Early proteins are encoded by the mRNAs which are synthesized prior to viral DNA synthesis. A number of the early proteins play a role in nucleotide metabolism and DNA synthesis in the infected cell. After the onset of viral DNA synthesis, the transcription of the late mRNAs is maximal and probably reflects a template abundancy requirement similar to that observed for
  • the late CMV proteins include the glycoprotein constituents of the viral envelope, the viral capsid proteins ar ⁇ other proteins which are necessary for assembly or structural integrity of the mature CMV particle and/or egress of the assembled virion from the infected cell.
  • examples of post-transcriptional controls are known to influence the appearance of some CMV proteins. Splicing of mRNAs is more common than observed in HSV gene expression and the nucleotide sequence
  • composition of the 5' nontranslated region in the cognate mRNA is reported to influence the synthesis of at least one early CMV protein.
  • the present invention is directed to an alternative approach to the treatment of such infections, the antisense inhibition of cytomegalovirus gene expression through the mediation of oligonucleotides or
  • Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA, or even double stranded DNA, such that the normal, essential functions of these intracellular nucleic acids are disrupted.
  • Hybridization is the sequence specific hydrogen bonding of oligonucleotides to Watson-Crick base pairs of RNA or single stranded DNA. Such base pairs are said to be complementary to one another.
  • hybridization arrest denotes a
  • Oligonucleotides Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton FL (1989) are two of the most
  • a second type of terminating event for antisense oligonucleotides involves enzymatic cleavage of the
  • RNA by intracellular RNase H.
  • the oligonucleotide or oligonucleotide analog which must be of the deoxyribo type, hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA.
  • Phosphorothioate oligonucleotides are a prominent example of an antisense agent which operates by this type of terminating event.
  • oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes.
  • Applications of oligonucleotides as diagnostics, research reagents, and potential therapeutic agents require that the
  • oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides towards nuclease degradation.
  • cytomegalovirus to inhibit the function of the messenger RNA.
  • cytomegalovirus through antisense interaction with messenger RNA of the virus.
  • Yet another object of this invention is to provide methods of diagnostics and therapeutics for
  • Novel oligonucleotides and oligonucleotide analogs are other objects of the invention.
  • cytomegalovirus RNA hybridizable with a selected sequence of a cytomegalovirus RNA. It has been determined that targeting cytomegalovirus mRNA coding for the IE1, IE2, or DNA polymerase proteins is a key to the effective antisense therapy with these oligonucleotides or oligonucleotide analogs.
  • oligonucleotides or oligonucleotide analogs either alone or in combination with a pharmaceutically acceptable carrier, to animals suspected of having
  • cytomegalovirus infections are provided.
  • RNA - - either its translation into protein, its translocation into the cytoplasm, or any other activity necessary to its overall biological function.
  • the failure of the RNA to perform all or part of its function results in failure of a portion of the genome controlling the norma life cycle of the virus.
  • oligonucleotides and analogs have between about 5 and about 50 nucleic acid base units. It is preferred that the oligonucleotide or analog be specifically hybridizable with mRNA coding for the CMV IE1, IE2, or DNA polymerase
  • the oligonucleotide analog may be modified to reduce nuclease resistance and to increase their efficacy.
  • the mRNA is interfered with to an extent ufficient to inhibit CMV replication.
  • oligonucleotide analogs which are capable of interacting with portions of CMV mRNA are comprehended. Animals suspected of having the disease are contacted with an oligonucleotide or oligonucleotide analog made in
  • the present invention is believed to be effective in the treatment of cytomegalovirus infections, either prophylactically or therapeutically.
  • Figure 1 is a bar graph showing the antiviral activity of oligonucleotides 2725 through 2890 against cytomegalovirus.
  • Figure 2 is a bar graph showing the antiviral activity of oligonucleotides 2891 through 3300 against cytomegalovirus.
  • Figure 3 is a line graph showing antiviral effects of eight oligonucleotides at doses from 0.01 to 10 ⁇ M .
  • Figure 4 is a line graph showing antiviral effects of three oligonucleotides at doses from 0.1 to 10 ⁇ M.
  • Antisense oligonucleotides hold great promise as therapeutic agents for the treatment of many human
  • Oligonucleotides specifically bind to the
  • oligonucleotides as a novel form of therapeutics.
  • an animal suspected of having a cytomegalovirus infection is treated by administering oligonucleotides or oligonucleotide analogs in accordance with this invention.
  • Persons of ordinary skill can easily determine optimum dosages, dosing met ⁇ r dologies and repetition rates. Such treatment is generally continued until either a cure is effected or a diminution in the disease state is achieved.
  • oligonucleotide refers to a polynucleotide formed from naturally occurring bases and pentofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits or their close hu.nologs.
  • Oligonucleotide analog refers to moieties which function similarly to oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotide analogs may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art. In accordance with some preferred embodiments. at least some of the phosphodiester bonds of the
  • oligonucleotide have been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA or DNA whose activity to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or
  • the phosphodiester bonds are substituted with other structures which are, at once, substantially non- ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
  • Oligonucleotide analogs may also include species which include at least some modified base forms.
  • purines and pyrimidines other than those normally found in nature may be so employed.
  • modifications on the pentofuranosyl portions of the nucleotide subunits may also occur as long as the essential tenets of this invention are adhered to.
  • cytomegalovirus RNA The oligonucleotides and
  • oligonucleotide analogs in accordance with this invention preferably comprise from about 3 to about 50 nucleic acid base units. It is more preferred that such
  • oligonucleotides and analogs comprise from about 8 to 25 nucleic acid base units, and still more preferred to have from about 12 to 25 nucleic acid base units.
  • a nucleic acid base unit is a base-sugar combination suitably bound to adjacent nucleic acid base unit through phosphodiester or other bonds.
  • messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5'-untranslated region, the 3'-untranslated region, and intron/exon junction ribonucleotides.
  • oligonucleotides and oligonucleotide analogs may be
  • oligonucleotide or analog is specifically hybridizable with a transcription initiation site, a translation initiation site, an intron/exon junction or sequences in the 5'- or 3'-untranslated region.
  • the HCMV genome is the most complex of the herpes viruses in terms of its genomic structure. Replication-defective mutants of HCMV have only been isolated for two viral genes, the immediate early complex (IE1 or IE2) and the DNA polymerase. These genes are known to play major roles in HCMV gene expression. They have been selected as primary targets for antisense compound design. Secondary target genes for the design of therapeutic antisense oligonucleotides and analogs have been selected by analogy to genes of herpes simplex virus. Such genes have been determined to be essential for herpes simplex virus replication and/or sensitive to antisense inhibition. Four gene products of herpes simplex virus which have recently shown to be sensitive to antisense inhibition are the virion tegument protein (UL48), the two proteins
  • ribonucleotide reductase enzyme UL39,40
  • virion phosphotransferase UL13
  • Other herpes simplex virus genes which are currently being studied are the auxiliary DNA replication enzymes (UL5, 8, 9, 29, 42, 52) and the major capsid protein (UL36) .
  • HCMV encodes proteins which have been identified as potentially
  • IE1 major immediate early transcript
  • the IE1 mRNA is an abundant RNA which is 1.9 kb in length and encodes a protein which migrates with an apparent molecular weight of 72 kDa on PAGE-SDS. This protein has been found in virions and controls the expression of itself as well as that of the IE2 gene product.
  • IE1 mRNA is synthesized by the cellular RNA polymerase.
  • IE2 mRNA A small amount of IE2 mRNA is made by processing of the IE1 mRNA during this early time of infection. Over time, levels of IE1 protein accumulate and bind the promoter region of the IE1 gene, repressing further transcription of the IE1 mRNA and allowing a weaker downstream promoter for the IE2 gene to control further synthesis of IE2 mRNA. It has been
  • the IE1 gene product may serve to boost viral transcription during a productive infection
  • the IE2 protein is capable of
  • the IE2 protein is believed to be one of the master switches for HCMV gene expression.
  • the other controlling switch of CMV genes is the DNA polymerase protein.
  • the selected targets within the mRNA sequences include regions of the mRNA which are known to control mRNA stability, processing and/or translational efficiency.
  • These target sites include the 5' cap regions and
  • nuc sig refers to nuclear localization signals of the IE2 protein.
  • RNA messenger RNA
  • oligonucleotides and analogs in accordance with the invention preferably have one of the foregoing sequences or an effective portion thereof.
  • nucleotides which persons of ordinary skill in the art can prepare from knowledge of the preferred antisense targets for the modulation of the viral infection.
  • oligonucleotides and oligonucleotide analogs of this invention can be used in diagnostics, therapeutics and as research reagents and kits.
  • the oligonucleotide or oligonucleotide analog is
  • oligonucleotides and oligonucleotide analogs of this invention in prophylaxis is also likely to be useful. Such may be accomplished, for example, by providing the medicament as a coating in condoms and the like. Use of pharmacologically acceptable carriers is also preferred for some embodiments.
  • the present invention is also useful in diagnostics and in research. Since the oligonucleotides and oligonucleotide analogs of this invention hybridize to nucleic acid from cytomegalovirus, sandwich and other assays can easily be constructed to exploit this fact.
  • oligonucleotide or analog with cytomegalovirus present in a sample suspected of containing it can routinely be
  • Such provision may include enzyme
  • Kits for detecting the presence or absence of cytomegalovirus may also be prepared.
  • Human foreskin fibroblast (ATCC #CRL 1635) cells used are obtained from the American Tissue Culture Collection. Cultures are grown in Dulbecco's
  • cytomegalovirus (HCMV strain AD169 or Towne) are grown on foreskin cells using low multiplicity infections
  • an infectious yield assay will be used.
  • foreskin cells are seeded at a density of 5 ⁇ 105 cells per well in Falcon 6 well tissue culture plates. Cells are overlaid with 2 ml of medium
  • Control wells receive 1 ml of medium which contains no oligonucleotide.
  • Virus is harvested into the overlay medium and triplicate wells of each experimental point are combined. The suspension is frozen at -80°C. Virus titer is
  • oligonucleotides which exhibit sequence complementarity to the CMV IE1, IE2 or DNA polymerase mRNAs will reduce the infectious yield of virus by 90%.
  • the mechanism of action of active CMV antisense compounds can also be validated.
  • the molecular nature of any mechanism of action study is dictated by the CMV gene sequence which is the target of oligonucleotide inhibition.
  • the most direct assays take advantage of the biological function of the protein encoded by the target CMV gene.
  • CMV genes which are amenable to these types of assays are the DNA polymerase and IE1 & 2 loci.
  • a simple mechanistic assay involves assessing the ability of target specific oligonucleotides to inhibit the incorporation of 3H-thymidine into viral DNA under conditions which favor viral DNA polymerase activity over cellular DNA polymerase activity.
  • the ability of the CMV IE proteins to inhibit the incorporation of 3H-thymidine into viral DNA under conditions which favor viral DNA polymerase activity over cellular DNA polymerase activity.
  • IE1 or IE2 responsive promoter regions are cloned 5' of an
  • oligonucleotide-induced changes in protein levels can be determined by immunoprecipitation of infected cell proteins, gel electrophoresis of the
  • Proteins of assayable biological activity can also be quantitated by immunoprecipitation and gel
  • CMV antisense drug will in a large degree depend on its ability to specifically interact with CMV RNA targets without adversely effecting host cell functions. Therefore it is important to evaluate the potential for nonspecific interactions and toxicities of active compounds. The potential for these adverse reactions is accessed in numerous models of acute and chronic
  • a therapeutic index for each active oligonucleotide compound is determined. Only those compounds exhibiting T.I. more than 100 are then considered for subsequent evaluation.
  • 2'-O-methyl phosphorothioate oligonucleotides were synthesized using 2'-O-methyl ⁇ -cyanoethyldiisopropyl- phosphoramidites (Chemgenes, Needham MA) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.
  • the 3'-base used to start the synthesis was a 2'-deoxyribonucleotide.
  • oligonucleotides were purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0.
  • Oligonucleotidedeoxynucleotides and their phosphorothioate analogs were judged from electrophoresis to be greater than 80% full length material.
  • ELISA assay for inhibition of HCMV replication by antisense oligonucleotides Oligonucleotides complementary to human cytomegalovirus mRNA were tested for antiviral activity in an ELISA-based assay of HCMV replication.
  • Normal human dermal fibroblasts (Clonetics Corp., San Diego CA) were grown in serum-free medium (Clonetics) and used to seed 96-well plates. When cells are approximately 80% confluent, they are pretreated with oligonucleotides. Approximately 20 hours after pretreatment the medium (containing
  • oligonucleotides is carefully poured off and the cells washed twice with warmed fibroblast basal medium (FBM, Clonetics). Cells are then infected with 100 ⁇ l/well of CMV stock diluted in FBM. The piates are incubated at 37°C for two hours. The medium (containing virus) is then carefully poured off and replaced with fresh, prewarmed FBM medium, 100 ⁇ l per well. The plates are briefly incubated at 37oC and then 5 ⁇ l of oligonucleotide, diluted in FBM, is reintroduced into the medium n each well. Two days later, cells are post-treated again with oligonucleotides in the same way. On day six, the plates are prepared for ELISA.
  • FBM warmed fibroblast basal medium
  • HCMV were tested for antiviral activity.
  • the sequences and gene targets for these oligonucleotides are presented in Table 2.
  • RNA transcribed from the major immediate early promoter of HCMV Since the two major protein products from this genomic region (IE1 and IE2) are synthesized from messenger RNA, which is
  • ISIS 2918 (SEQ ID NO: 18)
  • ISIS 2919 (SEQ ID NO: 19)
  • ISIS 2922 (SEQ ID NO: 22)
  • ISIS 2882 (SEQ ID NO: 12)
  • ISIS 3300 contains 2'-O-methyl-modified nucleosides with phosphorothioate linkages. This double modification was shown to convey much stronger antiviral activity upon the oligonucleotide than either the
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Abstract

Compositions and methods for modulating the effects of cytomegalovirus (CMV) infections are disclosed, comprising contacting CMV mRNA with an oligonucleotide or oligonucleotide analog which can bind with at least portions of the CMV RNA. In accordance with the preferred embodiments, oligonucleotides or oligonucleotide analogs are designed to bind with portions of the CMV mRNAs which code for the IE1, IE2 or DNA polymerase proteins. In accordance with a preferred embodiment, methods of treatment of human cytomegalovirus are disclosed.

Description

OLIGONUCLEOTIDES FOR MODULATING THE EFFECTS OF
CYTOMEGALOVIRUS INFECTIONS
FIELD OF THE INVENTION
This invention relates to the design and synthesis of antisense oligonucleotides which can be administered to inhibit the replication of cytomegalovirus and treat cytomegalovirus infections. These compounds can be used either prophylactically or therapeutically to reduce the severity of disease caused by cytomegaloviruses. Oligonucleotides and oligonucleotide analogs which are specifically hybridizable with RNA targets are described. BACKGROUND OF THE INVENTION
Cytomegaloviruses (CMV's) are ubiquitous in nature and are the most common causes of intrauterine infection. Congenital infection is common in newborns of infected mothers. In some populations, as much as 10% of children display perinatal infections. In a small
percentage of newborns, the infection is virulent,
involving multiple organs. Pronounced involvement of the reticuloendothelial and central nervous system is typical; and the inf tion is a major cause of mental retardation. Careful testing demonstrates that as many as 50% of
severely, prenatally infected adults may display
neuropsychiatric disease or deafness. Although extraneural organs are usually spared chronic morbidity, the virus can be detected in the kidney for years.
In the adult, cytomegalovirus-induced
mononucleosis is a lingering illness that causes
significant morbidity. If it occurs in immunosuppressed patients, the disease is more severe, and it may be
complicated by other infectious pathogens which may be fatal. Cytomegalovirus retinitis is a severe problem in immunosuppressed patients that often leads to blindness. Immunosuppressed patients are also very susceptible to CMV pneumonitis, which is one of the most lethal of human viral diseases. Although cytomegalovirus may play a role in the progression of HIV infection to AIDS by stimulating the transcription of the HIV long terminal repeats (LTR) in non-transformed co-infected T cells, histologic examination of adrenals and brains from AIDS patients has suggested that the adrenalitis, encephalitis and peripheral
neuropathy were caused by CMV infection.
CMV is considered to be an oncogenic virus. In vitro, CMV can transform cells and stimulate growth. Both human and non-human cells can undergo transformation when incubated with CMV. Transformed cells contain CMV antigens that are oncogenic when inoculated into appropriate
animals. Moreover, oncogenic potential has been associated with specific segments of the CMV genome.
Human CMV is a large, enveloped herpesvirus whose genome consists of a double-stranded DNA molecule which is approximately 240,000 nucleotides in length. This genome is the most complex of all DNA viruses and is approximately 50% larger than the genome of herpes simplex virus (HSV). Intact viral DNA is composed of contiguous long (L) and short (S) segments, each of which contains regions of unique DNA sequence flanked by homologous regions of repetitive sequence. As a group, the human CMV isolates share at least 80% sequence homology, making it nearly impossible to classify cytomegaloviruses into subgroups or subtypes, although variations in the restriction
endonuclease patterns of various CMV DNA preparations are identifiable in epidemiologically unrelated strains. The DNA of the prototypic strain of CMV (AD 169) has been sequenced and reported to contain a conservative estimate of 175 unique translational open reading frames (ORFs). A number of the predicted CMV gene products show homology to other human herpesvirus gene products. At least 42 ORFs encode putative glycoproteins and several of the CMV ORFs putatively encode proteins with amino acid homology to human opsin receptor proteins.
In permissive human fibroblasts, CMV gene
expression is regulated by a cascade of genetic events that act at both the transcriptional and translational levels. CMV gene expression can be divided into three phases which resemble those of HSV defined as the immediate early (IE), early and late periods. Following adsorption, penetration and uncoating of the virus, a group of viral transcripts, immediate early messenger RNAs (IE mRNAs) are synthesized within 1-4 hours even in the presence of translational inhibitors such as cycloheximide. In the normal course of infection, the IE mRNAs are translated and their protein products are instrumental in the onset of early
transcriptional events. At least 4 proteins are
synthesized from IE mRNAs; of these, one is a glycoprotein. The IE1 and IE2 proteins are transcriptional activating factors for other CMV genes and the IE3 protein encompasses a region of the CMV genome which can transform NIH 3T3 cells in vitro. Early proteins are encoded by the mRNAs which are synthesized prior to viral DNA synthesis. A number of the early proteins play a role in nucleotide metabolism and DNA synthesis in the infected cell. After the onset of viral DNA synthesis, the transcription of the late mRNAs is maximal and probably reflects a template abundancy requirement similar to that observed for
analogous HSV mRNAs. The late CMV proteins include the glycoprotein constituents of the viral envelope, the viral capsid proteins ar ι other proteins which are necessary for assembly or structural integrity of the mature CMV particle and/or egress of the assembled virion from the infected cell. In addition to the transcriptional controls operant upon CMV gene expression, examples of post-transcriptional controls are known to influence the appearance of some CMV proteins. Splicing of mRNAs is more common than observed in HSV gene expression and the nucleotide sequence
composition of the 5' nontranslated region in the cognate mRNA is reported to influence the synthesis of at least one early CMV protein.
Effective therapy for CMV has not yet been developed despite studies on a number of antivirals.
Interferon, transfer factor, adenine arabinoside (Ara-A), acycloguanosine (Acyclovir, ACV) and certain combinations of these drugs have been ineffective in controlling CMV infection. Based on preclinical and clinical data,
foscarnet (PFA) and ganciclovir (DHPG) show limited
potential as antiviral agents. PFA treatment has resulted in the resolution of CMV retinitis in five AIDS patients. DHPG studies have shown efficacy against CMV retinitis or colitis. DHPG seems to be well tolerated by treated individuals, but the appearance of a reversible
neutropenia, the emergence of resistant strains of CMV upon long-term administration, and the lack of efficacy against CMV pneumonitis limit the long term applications of this compound. The development of more effective and lesstoxic therapeutic compounds and methods is needed for both acute and chronic use.
Classical therapeutics has generally focused upon interactions with proteins in efforts to moderate their disease causing or disease potentiating functions. Such therapeutic approaches have failed for cytomegalovirus infections. The present invention is directed to an alternative approach to the treatment of such infections, the antisense inhibition of cytomegalovirus gene expression through the mediation of oligonucleotides or
oligonucleotide analogs.
Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA, or even double stranded DNA, such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides to Watson-Crick base pairs of RNA or single stranded DNA. Such base pairs are said to be complementary to one another.
The events which disrupt nucleic acid function are discussed by Cohen in Oligonucleotides: Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton FL, (1989) who proposes two possible types of terminating events. The first, hybridization arrest, denotes a
terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl
phosphonate oligonucleotides; P. S. Miller & P.O.P. Ts'O, Anti-Cancer Drug Design , Vol. 2, pp. 117-128 (1987); and α-anomer oligonucleotides, Cohen J.S. ed.,
Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton FL (1989) are two of the most
extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
A second type of terminating event for antisense oligonucleotides involves enzymatic cleavage of the
targeted RNA by intracellular RNase H. The oligonucleotide or oligonucleotide analog, which must be of the deoxyribo type, hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA.
Phosphorothioate oligonucleotides are a prominent example of an antisense agent which operates by this type of terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes. Applications of oligonucleotides as diagnostics, research reagents, and potential therapeutic agents require that the
oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides towards nuclease degradation.
Oligonucleotides and analogs modified to exhibit resistance to Tiucleases, to activate the RNase H
terminating event, and to hybridize with appropriate strength and fidelity to targeted RNA (or DNA) are greatly desired for antisense oligonucleotide diagnostics,
therapeutics and research with cytomegaloviruses.
OBJECTS OF THE INVENTION
It is an object of this invention to provide oligonucleotides and oligonucleotide analogs which are capable of hybridizing with messenger RNA of
cytomegalovirus to inhibit the function of the messenger RNA.
It is a further object to provide
oligonucleotides and analogs which can modulate the
expression of cytomegalovirus through antisense interaction with messenger RNA of the virus.
Yet another object of this invention is to provide methods of diagnostics and therapeutics for
cytomegalovirus in animals.
Methods, materials and kits for detecting the presence or absence of cytomegalovirus in a sample
suspected of containing it are further objects of the invention.
Novel oligonucleotides and oligonucleotide analogs are other objects of the invention.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the instant specification and appended claims.
SUMMARY OF THE INVENTION
In accordance with the present invention, methods of modulating the effects of cytomegalovirus infection are provided. Oligonucleotides and oligonucleotide analogs having a sequence of nucleotide bases specifically
hybridizable with a selected sequence of a cytomegalovirus RNA are provided. It has been determined that targeting cytomegalovirus mRNA coding for the IE1, IE2, or DNA polymerase proteins is a key to the effective antisense therapy with these oligonucleotides or oligonucleotide analogs. Methods for treating disease-states by
administering oligonucleotides or oligonucleotide analogs, either alone or in combination with a pharmaceutically acceptable carrier, to animals suspected of having
cytomegalovirus infections are provided.
This relationship is commonly denoted as
"antisense." The oligonucleotides and oligonucleotide analogs are able to inhibit the function of RNA - - either its translation into protein, its translocation into the cytoplasm, or any other activity necessary to its overall biological function. The failure of the RNA to perform all or part of its function results in failure of a portion of the genome controlling the norma life cycle of the virus.
It has now been found that oligonucleotides or oligonucleotide analogs can be designed especially for cytomegalovirus infections which are effective in
diminishing the infection. It is preferred that
oligonucleotides and analogs have between about 5 and about 50 nucleic acid base units. It is preferred that the oligonucleotide or analog be specifically hybridizable with mRNA coding for the CMV IE1, IE2, or DNA polymerase
proteins. The oligonucleotide analog may be modified to reduce nuclease resistance and to increase their efficacy.
In accordance with preferred embodiments, the mRNA is interfered with to an extent ufficient to inhibit CMV replication. Thus, oligonucleotides and
oligonucleotide analogs which are capable of interacting with portions of CMV mRNA are comprehended. Animals suspected of having the disease are contacted with an oligonucleotide or oligonucleotide analog made in
accordance with this invention. In particular, the present invention is believed to be effective in the treatment of cytomegalovirus infections, either prophylactically or therapeutically.
DESCRIPTION OF THE FIGURES
Figure 1 is a bar graph showing the antiviral activity of oligonucleotides 2725 through 2890 against cytomegalovirus.
Figure 2 is a bar graph showing the antiviral activity of oligonucleotides 2891 through 3300 against cytomegalovirus.
Figure 3 is a line graph showing antiviral effects of eight oligonucleotides at doses from 0.01 to 10μM .
Figure 4 is a line graph showing antiviral effects of three oligonucleotides at doses from 0.1 to 10 μM.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Antisense oligonucleotides hold great promise as therapeutic agents for the treatment of many human
diseases. Oligonucleotides specifically bind to the
complementary sequence of either pre-mRNA or mature mRNA, as defined by Watson-Crick base pairing, inhibiting the flow of genetic information from DNA to protein. Numerous recent studies have documented the utility of antisense oligonucleotides as biochemical tools for studying target proteins. Rothenberg et. al., J. Natl . Cancer Inst.
81:1539-1544 (1989); Zon, G. Pharmaceutical Res. , 5:539-549 1987). Because of recent advances in oligonucleotide chemistry, synthesis of nuclease-resistant
oligonucleotides, and availability of types of
oligonucleotide analogs which exhibit enhanced cell uptake, it is now possible to consider the use of antisense
oligonucleotides as a novel form of therapeutics.
For therapeutics, an animal suspected of having a cytomegalovirus infection is treated by administering oligonucleotides or oligonucleotide analogs in accordance with this invention. Persons of ordinary skill can easily determine optimum dosages, dosing metϋr dologies and repetition rates. Such treatment is generally continued until either a cure is effected or a diminution in the disease state is achieved.
It is to be expected that differences in the DNA of cytomegalovirus from different species and from
different types within a species exist. Thus, it is believed, for example, that the regions of the various cytomegalovirus species serve essentially the same function for the respective species and that interference with expression of the genetic information will afford similar results in the various species. This is believed to be so even though differences in the nucleotide sequences among the species doubtless exist.
Accordingly, nucleotide sequences set forth in the present specification will be understood to be
representational for the particular species being
described. Homologous or analogous sequences for different species of cytomegalovirus are specifically contemplated as being within the scope of this invention.
The present invention employs oligonucleotides and oligonucleotide analogs for use in antisense inhibition of the function of cytomegalovirus RNA. In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring bases and pentofuranosyl groups joined by native phosphodiester bonds. This term effectively refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits or their close hu.nologs.
"Oligonucleotide analog," as that term is used in connection with this invention, refers to moieties which function similarly to oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotide analogs may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art. In accordance with some preferred embodiments. at least some of the phosphodiester bonds of the
oligonucleotide have been substituted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA or DNA whose activity to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or
cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with other structures which are, at once, substantially non- ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
Oligonucleotide analogs may also include species which include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the pentofuranosyl portions of the nucleotide subunits may also occur as long as the essential tenets of this invention are adhered to.
Such analogs are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines), but which have one or more differences from natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with
cytomegalovirus RNA. The oligonucleotides and
oligonucleotide analogs in accordance with this invention preferably comprise from about 3 to about 50 nucleic acid base units. It is more preferred that such
oligonucleotides and analogs comprise from about 8 to 25 nucleic acid base units, and still more preferred to have from about 12 to 25 nucleic acid base units. As will be appreciated, a nucleic acid base unit is a base-sugar combination suitably bound to adjacent nucleic acid base unit through phosphodiester or other bonds. The oligonucleotides and analogs used in
accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed, however the actual synthesis of the oligonucleotides are well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotide analogs such as the phosphorothioates and alkylated derivatives.
In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5'-untranslated region, the 3'-untranslated region, and intron/exon junction ribonucleotides. Thus,
oligonucleotides and oligonucleotide analogs may be
formulated in accordance with this invention which are targeted wholly or in part to these associated
ribonucleotides as well as to the informational
ribonucleotides. In preferred embodiments, the
oligonucleotide or analog is specifically hybridizable with a transcription initiation site, a translation initiation site, an intron/exon junction or sequences in the 5'- or 3'-untranslated region.
The HCMV genome is the most complex of the herpes viruses in terms of its genomic structure. Replication-defective mutants of HCMV have only been isolated for two viral genes, the immediate early complex (IE1 or IE2) and the DNA polymerase. These genes are known to play major roles in HCMV gene expression. They have been selected as primary targets for antisense compound design. Secondary target genes for the design of therapeutic antisense oligonucleotides and analogs have been selected by analogy to genes of herpes simplex virus. Such genes have been determined to be essential for herpes simplex virus replication and/or sensitive to antisense inhibition. Four gene products of herpes simplex virus which have recently shown to be sensitive to antisense inhibition are the virion tegument protein (UL48), the two proteins
constituting the ribonucleotide reductase enzyme (UL39,40) and a virion phosphotransferase (UL13). Other herpes simplex virus genes which are currently being studied are the auxiliary DNA replication enzymes (UL5, 8, 9, 29, 42, 52) and the major capsid protein (UL36) . HCMV encodes proteins which have been identified as potentially
analogous in function to each of these herpes simplex virus proteins; these genes have been selected to serve as secondary targets in connection with this invention.
The molecular biology of immediate early transcription in HCMV has been as well elucidated as that of any transcriptional unit in the eucaryotic cell.
Briefly, synthesis of the major immediate early transcript (IE1) is controlled by a number of repeat units 51 of the mRNA cap site. These repeats are responsive to a number of transcriptional response molecules known to operate in cell-specific and differentiation specific manners. The IE1 mRNA is an abundant RNA which is 1.9 kb in length and encodes a protein which migrates with an apparent molecular weight of 72 kDa on PAGE-SDS. This protein has been found in virions and controls the expression of itself as well as that of the IE2 gene product. At the initial phase of immediate early transcription, only IE1 mRNA is synthesized by the cellular RNA polymerase. A small amount of IE2 mRNA is made by processing of the IE1 mRNA during this early time of infection. Over time, levels of IE1 protein accumulate and bind the promoter region of the IE1 gene, repressing further transcription of the IE1 mRNA and allowing a weaker downstream promoter for the IE2 gene to control further synthesis of IE2 mRNA. It has been
proposed that the IE1 gene product may serve to boost viral transcription during a productive infection and
alternatively to activate viral gene expression from the latent state. The observation of cell-type and
differentiation or hormonal responsive elements in the promoter of the IE1 gene are consistent with this
proposition. The IE2 protein is capable of
transcriptionally activating many of the HCMV early and late genes in a manner similar to other known
transactivating proteins of cellular and viral origin.
Thus, the IE2 protein is believed to be one of the master switches for HCMV gene expression. The other controlling switch of CMV genes is the DNA polymerase protein.
Transcription of the late viral genes operates at very low levels until the onset of viral DNA replication, after which the late genes are activated by an increased template availability. The exact molecular condition which is operant in this enhanced template availability is unclear, but the presence of the viral DNA polymerase and
replication of the genome are essential requirements for the observed effect.
The selected targets within the mRNA sequences include regions of the mRNA which are known to control mRNA stability, processing and/or translational efficiency.
These target sites include the 5' cap regions and
translation initiation control regions. The target
sequences for the IE1, IE2, and DNA polymerase genes are set forth in Table 1:
TABLE 1
TARGET SEQUENCES FOR CYTOMEGALOVIRUS
Oligonucleotide SYNTHESIS
TARGET GENE TARGET REGION TARGET DNA SEQUENCE
DNA POLYMERASE mRNA CAP SITE GGACCGGGACCACCGTCGTC
DNA POLYMERASE AUG REGION GTCCGCTATGTTTTTCAACCC DNA POLYMERASE CONSERVED AA
(717-732) CCTTCCATCATCATGGCCCAC
DNA POLYMERASE CONSERVED AA
(905-914) GGCGCGGGTCATCTACGGGAC
DNA POLYMERASE CMV INSERTION CCGCTGTGCCCGGCGACGCGG
(608-697) CCGCCCTTGCAATCTGCGCCG
GGCGTTTCACCCGGCTCCGGC
DNA POLYMERASE (1109-1159) GCGCCCGGTGTCCGGACGGCG
CCGCCGGCGTGGTTTCCCGGT CCGGCAAAGAAGAGGGCGCGG
IE1 mRNA CAP SITE GTGAACCGTCAGATCGCCTGG
IE1 AUG REGION CTTGACACGATGGAGTCCTC
IE1 I/E-1 GCCAAGAGTGACGTAAGTACC
IE1 I/E-2 GTCTTTTCTGCAGTCACCGTC
IE1 I/E-3 CAAGGTGCCACGGTACGTGTC
IE1 I/E-4 CATGTGTTTAGGCCCGAGAC
IE1 I/E-5 GGCAGAACTCGGTAAGTCTG
IE1 I/E-6 CCTCCTCTACAGTCAAACAG IE2 AUG/CAP SITE GCGCCTATCATGCTGCCCCTC
IE2 AUG REGION GCTCTCCCAGATGAACCACCC
IE2 I/E-1 CAAGATTGACGAGGTGAGCCG
IE2 I/E-2 CCCAAACAGGTCATGGTGCGC
IE2 NUC SIG-1 GCGTAAGAAACCGCGCAAAAC
IE2 NUC SIG-2 CGCAAGAAGAAGAGCAAACGC
In Table 1, the abbreviation I/E refers to the intron/exon junction while the AUG region is the
translation initiation region of IE2 mRNA whose
transcription is controlled by the IE2 specific promoter region. The abbreviation "nuc sig" refers to nuclear localization signals of the IE2 protein.
Oligonucleotides or analogs useful in the
invention are complementary to the DNA (especially for oligonucleotides directed to intron/exon junctions) or to the corresponding messenger RNA (mRNA) or pre-messenger
RNA. Thus, the oligonucleotides and analogs in accordance with the invention preferably have one of the foregoing sequences or an effective portion thereof. Thus, it is preferred to employ any of these oligonucleotides (or their analogs) as set forth above or any of the similar
nucleotides which persons of ordinary skill in the art can prepare from knowledge of the preferred antisense targets for the modulation of the viral infection.
The oligonucleotides and oligonucleotide analogs of this invention can be used in diagnostics, therapeutics and as research reagents and kits. For therapeutic use, the oligonucleotide or oligonucleotide analog is
administered to an animal suffering from a cytomegalovirus infection. It is generally preferred to apply the
therapeutic agent in accordance with this invention
internally such as intravenously, transdermally or
intramuscularly. Other forms of administration such as topically or intralesionally may also be useful. Inclusion in suppositories is presently believed to be likely to be useful. Use of the oligonucleotides and oligonucleotide analogs of this invention in prophylaxis is also likely to be useful. Such may be accomplished, for example, by providing the medicament as a coating in condoms and the like. Use of pharmacologically acceptable carriers is also preferred for some embodiments.
The present invention is also useful in diagnostics and in research. Since the oligonucleotides and oligonucleotide analogs of this invention hybridize to nucleic acid from cytomegalovirus, sandwich and other assays can easily be constructed to exploit this fact.
Provision of means for detecting hybridization of
oligonucleotide or analog with cytomegalovirus present in a sample suspected of containing it can routinely be
accomplished. Such provision may include enzyme
conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of cytomegalovirus may also be prepared.
EXAMPLES EXAMPLE 1
Cells and Virus: Human foreskin fibroblast (ATCC #CRL 1635) cells used are obtained from the American Tissue Culture Collection. Cultures are grown in Dulbecco's
Modified Eagle's Medium with 4.5 g/L glucose (high glucose DMEM) and supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 micrograms/ml) and L-glutamine (2 mM) . Stock cultures of human
cytomegalovirus (HCMV strain AD169 or Towne) are grown on foreskin cells using low multiplicity infections
(multiplicity of infection [MOI]=0.02 plaque forming units [PFU]/cell).
To assess the ability of oligonucleotides to inhibit CMV replication, an infectious yield assay will be used. To perform this assay, foreskin cells are seeded at a density of 5 × 105 cells per well in Falcon 6 well tissue culture plates. Cells are overlaid with 2 ml of medium
(high glucose DMEM with 10% FBS) and incubated at 37°C for 18-24 hours. Where appropriate, cells are overlaid with oligonucleotide preparations in 1 ml of medium at 24 hours after seeding the plates. Following an 18 hour incubation, all wells are rinsed with phosphate buffered saline and infected with HCMV at varying MOIs suspended in 0.5 ml of serum-free hgh glucose DMEM. Virus and cells are incubated at 37ºC for 90 minutes on a rocking platform. Following viral adsorption, unadsorbed virus is rinsed away by washing with phosphate buffered saline. Where appropriate, 1 ml of medium (high glucose DMEM with 10% FBS) containing 10 μM concentrations of oligonucleotide are added to the well and the cells are incubated for 4-5 days at 37°C.
Control wells receive 1 ml of medium which contains no oligonucleotide.
Virus is harvested into the overlay medium and triplicate wells of each experimental point are combined. The suspension is frozen at -80°C. Virus titer is
determined for each sample by plaque assay on human
foreskin cell monolayers. Dilutions of each virus
preparation are prepared and duplicate aliquots of each dilution are absorbed onto foreskin cells for 90 minutes with rocking. After adsorption, the unadsorbed virus inoculum is removed by rinsing the plates with phosphate buffered saline and the cells are overlaid with 2 ml of high glucose DMEM containing 5% FBS and 0.75% methyl cellulose. Cells are incubated at 37ºC for 12-14 days before plaques are fixed with formalin, stained with crystal violet and counted. Plaque counts from treated wells are compared with those from the control wells to establish the degree of inhibition of infectious virus production.
It is anticipated that treatment of CMV-infected cells with 10 μM concentrations of phophorothioate
oligonucleotides which exhibit sequence complementarity to the CMV IE1, IE2 or DNA polymerase mRNAs will reduce the infectious yield of virus by 90%.
EXAMPLE 2
The mechanism of action of active CMV antisense compounds can also be validated. The molecular nature of any mechanism of action study is dictated by the CMV gene sequence which is the target of oligonucleotide inhibition. The most direct assays take advantage of the biological function of the protein encoded by the target CMV gene.
The biological activity of an enzymatic protein often amplifies the end signal of such an assay so that the assay is very sensitive to even small changes in viral protein levels. Examples of CMV genes which are amenable to these types of assays are the DNA polymerase and IE1 & 2 loci.
For the DNA polymerase protein, a simple mechanistic assay involves assessing the ability of target specific oligonucleotides to inhibit the incorporation of 3H-thymidine into viral DNA under conditions which favor viral DNA polymerase activity over cellular DNA polymerase activity. The ability of the CMV IE proteins to
transactivate RNA synthesis of certain CMV genes has been used to devise a transient gene expression assay, whose activity depends upon the presence of biologically active IE1 or IE2 proteins in an infected cell. Briefly, IE1 or IE2 responsive promoter regions are cloned 5' of an
indicator gene (e.g., bacterial chloramphenicol acetyl transferase, CAT) in a plasmid vector. mhe vector is introduced into human foreskin cells, which in turn are infected with HCMV. The detection of CAT activity can be determined from cell lysates and CAT activity levels used to indirectly quantitate IE1 or IE2 protein levels. The effect of oligonucleotides on the CAT activity will be compared for both the IE1 and IE2 responsive constructs. In cases in which an overt biological activity is not easily demonstrable, oligonucleotide-induced changes in protein levels can be determined by immunoprecipitation of infected cell proteins, gel electrophoresis of the
immunoprecipitate in an SDS-acrylamide matrix, and
detection of target protein levels by autoradiography of the gel. Proteins of assayable biological activity can also be quantitated by immunoprecipitation and gel
electrophoretic techniques.
EXAMPLE 3
The value of a CMV antisense drug will in a large degree depend on its ability to specifically interact with CMV RNA targets without adversely effecting host cell functions. Therefore it is important to evaluate the potential for nonspecific interactions and toxicities of active compounds. The potential for these adverse reactions is accessed in numerous models of acute and chronic
cellular toxicity. Initially, active compounds are
evaluated for toxicity in infected human foreskin cells using 3H-leucine and 3H-thymidine to measure effects on protein and DNA synthesis, respectively. From
determinations of the oligonucleotide LD50 in these assays and the ID50 activity values obtained in the primary and secondary activity screens, a therapeutic index (T.I.) for each active oligonucleotide compound is determined. Only those compounds exhibiting T.I. more than 100 are then considered for subsequent evaluation.
EXAMPLE 4
Synthesis and characterization of oligonucleotides and analogs: Unmodified DNA oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine, β-cyanoethyldiisopropyl- phosphoramidites were purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-l,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step.
2'-O-methyl phosphorothioate oligonucleotides were synthesized using 2'-O-methyl β-cyanoethyldiisopropyl- phosphoramidites (Chemgenes, Needham MA) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. The 3'-base used to start the synthesis was a 2'-deoxyribonucleotide.
After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55ºC for 18 hours, the
oligonucleotides were purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0.
Oligonucleotidedeoxynucleotides and their phosphorothioate analogs were judged from electrophoresis to be greater than 80% full length material.
EXAMPLE 5
ELISA assay for inhibition of HCMV replication by antisense oligonucleotides: Oligonucleotides complementary to human cytomegalovirus mRNA were tested for antiviral activity in an ELISA-based assay of HCMV replication. Normal human dermal fibroblasts (Clonetics Corp., San Diego CA) were grown in serum-free medium (Clonetics) and used to seed 96-well plates. When cells are approximately 80% confluent, they are pretreated with oligonucleotides. Approximately 20 hours after pretreatment the medium (containing
oligonucleotides) is carefully poured off and the cells washed twice with warmed fibroblast basal medium (FBM, Clonetics). Cells are then infected with 100 μl/well of CMV stock diluted in FBM. The piates are incubated at 37°C for two hours. The medium (containing virus) is then carefully poured off and replaced with fresh, prewarmed FBM medium, 100μl per well. The plates are briefly incubated at 37ºC and then 5 μl of oligonucleotide, diluted in FBM, is reintroduced into the medium n each well. Two days later, cells are post-treated again with oligonucleotides in the same way. On day six, the plates are prepared for ELISA.
In preparation for ELISA, the medium is carefully poured off the plates, and cells are fixed in 200 μl of absolute ethanol per well. Cells are fixed for 30 minutes at room temperature, then ethanol is removed and plates are air-dried. Plates are blocked for one hour prior to ELISA with PBS containing 2% BSA. Blocking solution is removed and 100 μl of an anti-CMV antibody, diluted 1:2000 in PBS with 1% BSA, is added. Cells are incubated in antibody for one hour at 37ºC and washed three times in PBS. The secondary antibody, biotinylated goat anti-mouse IgG
(Bethesda Research Labs, MD) , is diluted 1:1000 in PBS with 1% BSA, and incubated with cells for one hour at 37°C.
Cells are then washed and incubated for one hour at 37ºC in streptavidin-B-D-galactosidase. Color is developed with chlorophenol red-B-D-galactopyranoside, 20 mg dissolved in 10 ml of 50 mM Na Phosphate, 1.5 mM MgCl2; plates are shaken for 10 minutes and the absorbance is read at 575 nm.
Twenty-four oligonucleotides complementary to
HCMV were tested for antiviral activity. The sequences and gene targets for these oligonucleotides are presented in Table 2.
TABLE 2
Oligonucleotide Analogs Tested for Activity Against HCMV
SEQ ID
NO ISIS # Nucleotide #s Target Sequence Type
1 2725 Nonsense GTG TCA AGT GGC ACC ATA CG P=S
2 2726 Nonsense TGG AAA GTG TAC ACA GGC GAA P=S
3 2728 80618-80639 DNA pol. AUG GGG TTG AAA AAC ATA GGG GAG P=S
4 2729 172755-172776 IE1 AUG GAG GAC TCC ATC GTG TCA AG P-S
5 2855 78445-78466 DNA pol. coding GTG GGC CAT GAT GAT GGA AGG P-S
6 2856 77903-77924 DNA pol. coding GTC CCG TAG ATG ACC CGC GCC P=S
7 2869 78688-78709 DNA pol. coding CGG CGC AGA TTG CAA GGG CGG P=S
8 2870 78655-78676 DNA pol. coding GCC GGA GCC GGG TGA AAC GCC P=S
9 2871 77305-77326 DNA pol. coding CGC CGT CCG GAC ACC GGG CGC P=S
10 2876 77250-77271 DNA pol. coding ACC GGG AAA CCA CGC CGG CGG P=S
11 2877 77155-77176 DNA pol. coding CCG CGC CCT CTT CTT TGC CGG P=S
12 2882 173601-173622 IE1 int/exon 1 GGT ACT TAC GTC ACT CTT GGC P=S
13 2883 172775-172796 IE1 irit/exon 2 GAC GGT GAC TGC AGA AAA GAC P=S
14 2884 172686-172707 IE1 int/exon 3 GAC ACG TAC CGT GGC ACC TTG P=S
15 2890 172572-172592 IE1 int/exon 4 GTC TCG GGC CTA AAC ACA TG P=S
16 2891 172387-172407 IE1 int/exon 5 CAG ACT TAC CGA CTT CTG CC P=S
17 2908 172218-172238 IE1 int/exon 6 CTG TTT GAC TGT AGA GGA GG P=S
18 2918 170373-170394 IE2 AUG GGG TCC TTC ATC TGG GAG AGC P=S
19 2919 170004-170025 IE2 int/exon 1 CGG CTC AGG TCG TCA ATC TTG P=S
20 2920 169535-169556 IE2 int/exon 2 GCG CAC CAT GAC CTG TTT GGG P=S
21 2921 170652-170673 IE2 nuc sig 1 GTT TTG CGC GGT TTC TTA CGC P=S
22 2922 170120-170141 IE2 nuc sig 2 GCG TTT GCT CTT CTT CTT GCG P=S
23 3245 173713-173734 IE1/IE2 5 'cap CGT CTC CAG GCG ATC TGA CGC P=S
24 3246 173710-173731 IE1/IE2 5 'cap TGG CGT CTC CAG GCG ATC TGA P=S
3258 2'-O-Me
3300 P-S/2'-O-Me
25 3224 Random TCT GAG TAG CAG AGG AGC TC P=S/2'-O-Me
26 3221 Random CTC CAC GCG AAT TTT AAC ACA P=S
3266 '' '' 2'-O-Me
27 1238 Random ACT CGG GCT GCC ACT TGA CAG P=S
Of the oligonucleotides tested, eight were complementary to mRNA encoding the HCMV DNA polymerase, and the remainder were complementary to RNA transcribed from the major immediate early promoter of HCMV. Since the two major protein products from this genomic region (IE1 and IE2) are synthesized from messenger RNA, which is
transcribed from a common promoter, eight of these
compounds are complementary to both the IE1 and IE2 mRNA. Three compound are complementary only to the IE1 and IE2 mRNA. Three compounds are complementary only to the IE1 mRNA, and the remaining five are specific for IE2 mRNA.
At a screening concentration of 5 μM all but one compound showed some reduction of viral replication
compared to untreated cells (Figures 1 and 2). Some compounds exhibited a markedly greater inhibition of virus replication than control oligonucleotides, and these were chosen for further characterization.
Dose-response experiments differentiated between non-specific effects and sequence-specific inhibition of HCMV replication by antisense oligonucleotides. Compounds ISIS 2922 (SEQ ID NO: 22), ISIS 2882 (SEQ ID NO: 12), ISIS 2918 (SEQ ID NO: 18), ISIS 2919 (SEQ ID NO: 19) and ISIS 3300 (SEQ ID NO: 24, P=S/2'-O-Me) all showed inhibition of HCMV replication at lower doses than randomized
oligonucleotides with no complementarity to HCMV (Figure
3). Compounds ISIS 2918 (SEQ ID NO: 18) , ISIS 2919 (SEQ ID NO: 19), and ISIS 2922 (SEQ ID NO: 22) are complementary to IE2 RNA sequences. ISIS 2882 (SEQ ID NO: 12) and ISIS 3300 (SEQ ID NO: 24, P=S and 2'-O-Me) are complementary to the 5' cap region of IE1 and IE2 transcripts. Except where indicated in Table 2, oligonucleotides used are
phosphorothioates; ISIS 3300 contains 2'-O-methyl-modified nucleosides with phosphorothioate linkages. This double modification was shown to convey much stronger antiviral activity upon the oligonucleotide than either the
phosphorothioate (ISIS 3246, moderate activity) or the 2'-O-methyl modification (ISIS 3258, slight activity) alone. The activity of ISIS 2919 and ISIS 2922 relative to a randomized control oligonucleotide was confirmed in an independent dose-response experiment (Figure 4).
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Anderson, Kevin P.
Draper, Kenneth G.
(ii) TITLE OF INVENTION: Oligonucleotides for
Modulating the Effects of Cytomegalovirus Infections
(iii) NUMBER OF SEQUENCES: 27
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Woodcock Washbum Kurtz Mackiewicz & Norris
(B) STREET: One Liberty Place - - 46th floor
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19103
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: WORDPERFECT 5.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a
(B) FILING DATE: herewith
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Licata, Jane M.
(B) REGISTRATION NUMBER: 32,257
(C) REFERENCE/DOCKET NUMBER: ISIS-0408 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568-3100
(B) TELEFAX: (215) 568-3439
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GTGTCAAGTG GCACCATACG 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TGGAAAGTGT ACACAGGCGA A 21
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GGGTTGAAAA ACATAGCGGA C 21
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI -SENSE : YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GAGGACTCCA TCGTGTCAAG 20
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GTGGGCCATG ATGATGGAAG G 21 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GTCCCGTAGA TGACCCGCGC C 21
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CGGCGCAGAT TGCAAGGGCG G 21
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GCCGGAGCCG GGTGAAACGC C 21
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CGCCGTCCGG ACACCGGGCG C 21
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE. CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
ACCGGGAAAC CACGCCGGCG G 21 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCGCGCCCTC TTCTTTGCCG G 21
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GGTACTTACG TCACTCTTGG C 21
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GACGGTGACT GCAGAAAAGA C 21
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GACACGTACC GTGGCACCTT G 21
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GTCTCGGGCC TAAACACATG 20
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETIAAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CAGACTTACC GACTTCTGCC 20 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
CTGTTTGACT GTAGAGGAGG 20 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GGGTCCTTCA TCTGGGAGAG C 21
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 19:
CGGCTCAGGT CGTCAATCTT G 21
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GCGCACCATG ACCTGTTTGG G 21
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
GTTTTGCGCG GTTTCTTACG C 21
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GCGTTTGCTC TTCTTCTTGC G 21
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
Ti>) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CGTCTCCAGG CGATCTGACG C 21 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
TGGCGTCTCC AGGCGATCTG A 21
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TCTGAGTAGC AGAGGAGCTC 20
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SLQUENCE DESCRIPTION: SEQ ID NO: 26:
CTCCACGCGA ATTTTAACAC A 21
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
ACTCGGGCTG CCACTTGACA G 21

Claims

CLAIMS What is claimed is
1. An oligonucleotide or oligonucleotide analog specifically hybridizable with at least a portion of RNA or DNA deriving from IE1, IE2 or DNA polymerase genes of a cytomegalovirus.
2. The oligonucleotide or oligonucleotide analog of claim 1 specifically hybridizable with at least a portion of the mRNA cap site, the AUG region, the conserved amino acid region, or the CMV insertion regions between bases 608-697 or 1109-1159 of the DNA polymerase gene.
3. The oligonucleotide or oligonucleotide analog of claim 1 specifically hybridizable with at least a portion of the mRNA cap site, the AUG region or an
intron/exon junction region of the IE1 gene.
4. The oligonucleotide or oligonucleotide analog of claim 1 specifically hybridizable with at least a portion of the AUG/CAP site, the AUG region, an IE2
specific intron/exon junction region, or a nuclear location signal region of the IE2 gene.
5. The oligonucleotide or oligonucleotide analog of claim 1 in a pharmaceutically acceptable carrier.
6. The oligonucleotide or oligonucleotide analog of claim 1 having from 5 to about 50 nucleic acid base units.
7. The oligonucleotide or oligonucleotide analog of claim 1 having from 8 to about 25 nucleic acid base units.
8. The oligonucleotide or oligonucleotide analog of claim 1 having from 12 to about 25 nucleic acid base units.
9. The oligonucleotide or oligonucleotide analog of claim 1 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise sulfur-containing species.
10. The oligonucleotide or oligonucleotide analog of claim 1 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise phosphorothioate moieties.
11. An oligonucleotide or oligonucleotide analog complementary to the DNA or corresponding RNA or pre-messenger RNA of at least a portion of one of the
sequences:
GGA CCG GGA CCA CCG TCG TC,
GTC CGC TAT GTT TTT CAA CCC,
CCT TCC ATC ATC ATG GCC CAC,
GGC GCG GGT CAT CTA CGG GAC,
CCG CTG TGC CCG GCG ACG CGG CCG CCC TTG CAA TCT GCG CCG GGC GTT TCA CCC GGC TCC GGC,
GCG CCC GGT GTC CGG ACG GCG CCG CCG GCG TGG TTT CCC GGT
CCG GCA AAG AAG AGG GCG CGG,
GTG AAC CGT CAG ATC GCC TGG,
CTT GAC ACG ATG GAG TCC TC,
GCC AAG AGT GAC GTA AGT ACC,
GTC TTT TCT GCA GTC ACC GTC,
CAA GGT GCC ACG GTA CGT GTC,
CAT GTG TTT AGG CCC GAG AC,
GGC AGA ACT CGG TAA GTC TG,
CCT CCT CTA CAG TCA AAC AG,
GCG CCT ATC ATG CTG CCC CTC,
GCT CTC CCA GAT GAA CCA CCC,
CAA GAT TGA CGA GGT GAG CCG,
CCC AAA CAG GTC ATG GTG CGC,
GCG TAA GAA ACC GCG CAA AAC, or
CGC AAG AAG AAG AGC AAA CGC.
12. The oligonucleotide or oligonucleotide analog of claim 11 in a pharmaceutically acceptable carrier.
13. The oligonucleotide or oligonucleotide analog of claim 11 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise sulfur-containing species.
14. The oligonucleotide or oligonucleotide analog of claim 11 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise phosphorothioate moieties.
15. A method for modulating the activity of a
cytomegalovirus infection comprising contacting an animal suspected of having a CMV infection with an oligonucleotide or oligonucleotide analog specifically hybridizable with at least a portion of RNA or DNA deriving from IE1, IE2 or DNA polymerase genes of the cytomegalovirus.
16. The method of claim 15 wherein said infection is by human cytomegalovirus.
17. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog is specifically hybridizable with at least a portion of the mRNA cap site, the AUG region, the conserved amino acid region, the CMV insertion regions between bases 608-697 or 1109-1159 of the DNA polymerase gene.
18. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog is specifically hybridizable with at least a portion of the mRNA cap site, the AUG region or an intron/exon junction region of the IE1 gene.
19. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog is specifically hybridizable with at least a portion of the AUG/CAP site, the AUG region, an IE2 specific intron/exon junction region, or a nuclear location signal region of the IE2 gene.
20. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog is in a
pharmaceutically acceptable carrier.
21. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog of claim 1 has from 5 to about 50 nucleic acid base units.
22. The method of claim 15 wherein the oligonucleotide or oligonucleotide analog of claim 1 has from 8 to about 25 nucleic acid base units.
23. The method of claim 15 wherein the
oligonucleotide or oligonucleotide analog of claim 1 has from 12 to about 25 nucleic acid base units.
24. The method of claim 15 wherein at least some of the linking groups between nucleotide units of the
oligonucleotide comprise sulfur-containing species.
25. The method of claim 15 wherein at least some of the linking groups between nucleotide units of the
oligonucleotide comprise phosphorothioate moieties.
26. A method for modulating the activity of a
cytomegalovirus infection comprising contacting an animal suspected of having a CMV infection with an oligonucleotide or oligonucleotide analog complementary to the DNA or corresponding RNA or pre-messenger RNA of at least a portion of one of the sequences:
GGA CCG GGA CCA CCG TCG TC,
GTC CGC TAT GTT TTT CAA CCC,
CCT TCC ATC ATC ATG GCC CAC,
GGC GCG GGT CAT CTA CGG GAC,
CCG CTG TGC CCG GCG ACG CGG CCG CCC TTG CAA TCT GCG CCG GGC GTT TCA CCC GGC TCC GGC,
GCG CCC GGT GTC CGG ACG GCG CCG CCG GCG TGG TTT CCC GGT CCG GCA AAG AAG AGG GCG CGG,
GTG AAC CGT CAG ATC GCC TGG,
CTT GAC ACG ATG GAG TCC TC,
GCC AAG AGT GAC GTA AGT ACC,
GTC TTT TCT GCA GTC ACC GTC,
CAA GGT GCC ACG GTA CGT GTC,
CAT GTG TTT AGG CCC GAG AC,
GGC AGA ACT CGG TAA GTC TG, CCT CCT CTA CAG TCA AAC AG,
GCG CCT ATC ATG CTG CCC CTC,
GCT CTC CCA GAT GAA CCA CCC,
CAA GAT TGA CGA GGT GAG CCG,
CCC AAA CAG GTC ATG GTG CGC,
GCG TAA GAA ACC GCG CAA AAC, or
CGC AAG AAG AAG AGC AAA CGC.
27. An oligonucleotide or oligonucleotide analog specifically hybridizable with the DNA or corresponding mRNA or pre-mRNA of cytomegalovirus comprising at least a portion of one of the sequences identified in Table 2.
28. The oligonucleotide or oligonucleotide analog of claim 27 in a pharmaceutically acceptable carrier.
29. The oligonucleotide or oligonucleotide analog of claim 27 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise sulfur-containing species.
30. The oligonucleotide or oligonucleotide analog of claim 27 wherein at least some of the linking groups between nucleotide units of the oligonucleotide comprise phosphorothioate moieties.
31. The oligonucleotide or oligonucleotide analog of claim 27 wherein at least some of the nucleotide bases have 2'-O-alkyl modifications.
32. The oligonucleotide or oligonucleotide analog of claim 31 wherein the modified bases are 2'-O-methyls.
33. A method for modulating the activity of a
cytomegalovirus infection comprising contacting an animal suspected of having a cytomegalovirus infection with a therapeutically effective amount of an oligonucleotide or oligonucleotide analog specifically hybridizable with the DNA or corresponding mRNA or pre-mRNA of cytomegalovirus comprising at least a portion of one of the sequences identified in Table 2.
34. The method of claim 33 wherein the
oligonucleotide or oligonucleotide analog is in a
pharmaceutically acceptable carrier.
35. The method of claim 33 wherein at least some of the linking groups between nucleotide units of the
oligonucleotide comprise sulfur-containing species.
36. The method of claim 33 wherein at least some of the linking groups between nucleotide units of the
oligonucleotide comprise phosphorothioate moieties.
37. The method of claim 33 wherein at least some of the nucleotide bases of the oligonucleotide have 2'-O-alkyl modifications.
38. The method of Claim 37 wherein the modified bases are 2'-O-methyls.
39. The oligonucleotide or oligonucleotide analog of claim 27 comprising 2'-O-methyl-modified nucleosides with phosphorothioate linkages.
40. The method of claim 33 wherein said
oligonucleotide comprises 2'-O-methyl-modified nucleosides with phosphorothioate linkages.
PCT/US1991/005815 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections WO1992003456A1 (en)

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DE69126710T DE69126710T2 (en) 1990-08-16 1991-08-14 OLIGONUCLEOTIDES FOR MODULATING THE EFFECTS OF CYTOMEGALOVIRUS INFECTIONS
CA002089666A CA2089666C (en) 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections
KR1019930700439A KR970005273B1 (en) 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections
US07/927,506 US5591720A (en) 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections
EP91914366A EP0544713B1 (en) 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections
AU84393/91A AU649717B2 (en) 1990-08-16 1991-08-14 Oligonucleotides for modulating the effects of cytomegalovirus infections
BR919106751A BR9106751A (en) 1990-08-16 1991-08-14 OLIGONUCLEOTIDE OR NUCLEOTIDE ANALOG AND PROCESS FOR MODULATING THE ACTIVITY OF A CITOMEGALOVIRUS INFECTION
NO93930515A NO930515L (en) 1990-08-16 1993-02-12 OLIGONUCLEOTIDES FOR MODULATING THE EFFECTS OF CYTOMEGALOVIRUS INFECTIONS
FI930658A FI930658A (en) 1990-08-16 1993-02-15 OLIGONUCLEOTIDER FOR MODULATION OF AV EFFECTS AV CYTOMEGALOVIRUS INFECTION
US08/233,711 US5595978A (en) 1990-08-16 1994-04-26 Composition and method for treatment of CMV retinites
GR970402519T GR3024873T3 (en) 1990-08-16 1997-09-26 Oligonucleotides for modulating the effects of cytomegalovirus infections

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